Thirty temperature-sensitive mutants of encephalomyocarditis (EMC) virus were previously isolated and partially characterized. Ten of these mutants have a similar defect in the proteolytic processing of the viral protein precursors when grown at the nonpermissive temperature of 39.5C. With an in vitro protein synthesis system, we have obtained results with some of the cleavage-defective mutants which indicate that they do not seem to have defective proteolytic cleavage enzymes, but appear to be defective in the site to be cleaved. We propose to examine all the cleavage-defective mutants that we have isolated to determine if they have a similar defect. We are also attempting to isolate mutants in the hypothetical cleavage enzyme. One cleavage-defective mutant appears to have a defect in the capside precursor protein epsilon. This defect appears to prevent the proper assembly of the subassembly structures of EMC virus resulting in an accumulation of some of these structures. We propose to also examine the maturation of other cleavage-defective mutants of EMC virus. We have begun to characterize EMC virus polypeptides with the two-dimensional gel electrophoresis method. Three of the four capsid polypeptides can be resolved with this method, which involves isolectric focusing in the first dimension and sodium dodecyl sulfate gel electrophoresis in the second dimension. Eight virion and nonvirion polypeptides made during infection by EMC virus can be resolved. Most of these polypeptides are stable protein products and are not precursor polypeptides. We propose to examine the viral polypeptides in mutant virus infected cells and in mutant virions with two-dimensional gel electrophoresis to determine if mutant viral proteins contain differences in charged amino acids relative to wild type viral proteins.